In multicellular organisms, genomic stability and integrity is maintained by the combined actions of an accurate DNA replication machinery and a complex network of DNA repair pathways. DNA joining is an essential step in DNA replication, in DNA excision repair and in the repair of DNA strand breaks. Genetic studies have demonstrated that the DNA ligase encoded by the human LIG4 gene and functionally homologous DNL4 gene of Saccharomyces cerevisiae is a key factor in the repair of DNA double-strand breaks (DSB)s by non- homologous end joining (NHEJ). In mammalian cells, NHEJ is required for genomic stability with defects in this pathway resulting in the type of genetic rearrangements frequently observed in cancer cells and an increased incidence of cancer in mouse models. In this proposal, we will focus on delineating the molecular mechanisms of DNA ligase IV-dependent NHEJ. Previous studies with yeast NHEJ factors have revealed functional interactions among yKu70/yKu80 (yKu), Mre11/Rad50/Xrs2 (MRX) and Dnl4/Lif1 complexes and defined how these factors are recruited to in vivo DNA double strand breaks. Our preliminary studies indicate that another key NHEJ factor, Nej1, participates at every step of the NHEJ pathway. This proposal will focus on elucidating the roles of Nej1 and its human ortholog XLF in NHEJ using a combination of in vitro and in vivo approaches. In Specific Aim 1, we will characterize the complexes formed by yKu, Nej1 and Dnl4/Li1 and functionally homologous human proteins, Ku, DNA ligase IV/XRCC4 and XLF, at DNA ends. In Specific Aim 2, we will elucidate the molecular mechanism of the ligation step that completes the repair of DSBs by NHEJ and determine how Nej1(XLF) contributes to this reaction. In Specific Aim 3, we will delineate the role of Nej1 in the end processing reactions of NHEJ. Because of the conservation of NHEJ factors among eukaryotes, we anticipate that results from our studies will provide insights into mechanisms and regulation of NHEJ in mammalian cells. The studies in this proposal will contribute to an overall picture of how the repair of DSBs by NHEJ prevents the deleterious genetic changes that drive cancer formation and progression. In addition, they will provide a framework for the identification of novel, specific modulators of the human NHEJ pathway that may have clinical utility as radiosensitizers or radioprotectors. PUBLIC HEALTH RELEVANCE: It is well established that genomic instability drives the progression of a normal cell into a cancer cell. Human cells have a complex network of pathways that act together to maintain genome stability. There are now many examples were studies in simpler model eukaryotes, such as the yeast Saccharomyces cerevisiae, have provided important insights into the mechanisms and regulation of the pathways that maintain genome stability in humans. A mechanistic understanding of these pathways will provide fundamental insights into tumor suppression. In addition, genomic instability is a characteristic of tumor cells, indicating that there are differences in the pathways that normally maintain stability. These differences between normal and cancer cells offer an opportunity to develop therapeutic strategies that selectively target cancer cells.